1) Manuel L. Gonzalez-Garay presented research projects at UTHealth from 2009-2015 investigating rare genetic disorders using next-generation sequencing and metabolomics. 2) An experimental design involved whole exome sequencing of 81 healthy volunteers from the Young Presidents' Organization to explore the practical value and challenges of genomic information for healthy individuals. 3) Analysis of the sequencing data and metabolomics profiles identified several disease-causing variants and metabolic deficiencies, demonstrating the potential for precision medicine approaches in volunteers of normal health.

1. Manuel L. Gonzalez-Garay, Ph.D.
Adjunct Professor, The Center for Research and
Development
in Health Sciences (CIDICS). Universidad
Autónoma de Nuevo León.
September 18, 2015
(Host: Dr. Steven Norris)
Progress toward Genomic and
Precision Medicine at UTHealth
CIDICS

2. Research projects at UTHealth (2009-2015)
Project Collaborator(s)
YPO Project (81 healthy individuals) Dr. Thomas Caskey
Genetics of Schizophrenia and Bipolar Drs. Caskey, Soares, Escamilla
Genetics of lymphedema Dr. Eva Sevick Ongoing 2 publications, 2 manuscripts
Genetics of Prader-Willi and Autism Drs. Schaaf & Beaudet
Dercum's disease
Familial multiple lipomatosis
Lipedema
Madelung’s disease
Dr. Karen L. Herbst (Tucson, AZ) Completed 1 manuscript,
transferred to UA
Rare genetic disorders
Inherited hearing disorders
Inherited eye disorders
Inherited panic disorders
Sporadic leucodystrophy
Actinic prurigo in Native Americans
• Dr. Felipe H. Sandoval-Avilés
• Dr. Miguel Valdés-Adamchik
• Dr. Ricardo Salinas-Ruiz
• Dr. Sergio A. Cuevas-Covarrubias
• Dr. Augusto Rojas-Martínez
• Dr. Rocío Ortiz-López
WGS Rats with hypertension and kidney failure Dr. Peter Doris Ongoing 2 Publications
WGS Induced pluripotential stem cells Dr. Brian Davis Completed 1 publication, 1 manuscript
Tuberous sclerosis, Spina bifida Drs. Hope Northrup & Au, Kit-Sing Completed pending
manuscripts
Differential expression of Candida sp during Infection Dr. Michael Lorenz Completed pending manuscript
Completed 3 publications
Completed 1 publications
Ongoing, transferred to UANL

3. Personalized Precision Medicine
99.93 % Similarity between humans
4 million genetic variants per person
(against the human template)
The use of an individual's genetic profile to guide decisions made in regard to the prevention,
diagnosis, and treatment of disease. Knowledge of a patient's genetic profile can help doctors
select the proper medication or therapy and administer it using the proper dose or regimen.

4. Bottleneck for precision medicine
1. Genetics and Genomic education for
patients and medical professionals
2. Standards for Genomic interpretation
3. Phenotype-Genotype
4. Instrumentation to detect and describe
new human phenotypes

5. Increasing number of aging adults
• We decided to explore the use of Next Generation Sequencing (NGS) to
provide genetic discoveries to adults
• What is the practical value of NGS information for healthy volunteers?
• What are the challenges and barriers to the adoption of this powerful
technology into medical practice?
HCM/DCM/ARVC STROKE ALZHEIMER PARKINSON Other disorders
Medicine treats pathologies

6. Experimental Design
• Genome Education Program (The Cullen Trust for Higher Education) 2009.
– Educate Patients and their physicians about genetics
• Young Presidents’ Organization YPO
– Requested a education program for their members for their annual competition
• Organized with Houston Chapter: 450 members.
• Education Conference Day: >150 attendees – Institute of Molecular
Medicine (IMM), UTHealth at Houston.
• IRB approved volunteer protocol “Need to know results”: 81 volunteers.
• Whole Exon sequencing.
• Medical report focused on causative alleles for Mendelian disorders.
• All participants require a face to face meeting with our genetic counselor
[CTC] (medical history and 3 generation pedigree)
• A second individual genetic counseling session to discuss the written
reports.

7. Difficulties of the project
Familial analysis vs single sample analysis
Unrelated individuals
Caskey & Gonzalez-Garay. Adult
Genetic Risk Screening.
ARM in press.

8. Detect real variants from false positive variants
Training set
Two orthogonal technologies
Illumina and Complete Genomics
FP
R
scikit-learn
Machine Learning Python
Support vector machine
Linear kernel
R
FP
Distance between variants
V
V
V
V
V
V
V E
E
EE
E
E
E
E
V

9. Workflow for processing NGS data
Annotated SNPS/Indels & remove FP with ML
SNPs
Indels
SAM files
Reference Sequence
NovoAlign mapping to reference after adapter stripping
GATK’s Bayesian
SNP/Indel caller
BAM files
•Remove duplicates
•Local realignments
•Recalibrate alignments
In-house algorithm,
snpEff &
ANNOVAR
GATK
Paired-end short reads
SAMtools/Picard
Gonzalez-Garay M.L., McGuire AL, Pereira S, & Caskey CT (2013)
Personalized genomic disease risk of volunteers. Proc Natl Acad Sci U S A.
110(42):16957-16962.
~300 bp
110 bp 110 bp
50 Million fragments
WES
VCF file

10. Pipeline to generate Variants Reports
YPOVariants Report
dbSNP 132
CCDS, refSeq, known Gene (UCSC)
snpEff (ns-coding variants)
Filter out variants with Internal Frequency filter >= 3%
Search in HGMD Db for Disease Causing Mutations
Filter out variants MAF >= 0.5%
Filter out variants have been consider benign
for at least 2/3 predictions tools
(Polyphen-2, Sift and MutationTaster)
Omim/Pubmed
Candidate Genes
Variant Call Format (VCF)
Gonzalez-Garay M.L., McGuire AL, Pereira S, & Caskey CT (2013)
Personalized genomic disease risk of volunteers. Proc Natl Acad Sci U S A.
110(42):16957-16962.
81 Healthy Volunteers
> 50 Yrs.

11. Example of a YPO report
Patient
Type
Type

12. Disorders
Affected
volunteers
Volunteers with
Positive Family
History
known mutations
found in
volunteers
(HGMD)
new mutations
in known Risk
genes found in
volunteers
Recessive 0 0 208 63
X-linked recessive 0 0 3 0
Breast Cancer 2 with new alleles 9 7 2
Colon Cancer 0 4 with known alleles 8 1
Other Cancer 4 with new alleles 2 with known alleles 11 9
Cardiomyopathy-affected 12 12 9 8
Cardiomyopathy-unaffected 0 11 5 8
Neurodegenerative 0 5 with known alleles 10 17
Summary of findings
Gonzalez-Garay M.L., McGuire AL, Pereira S, & Caskey CT (2013)
Personalized genomic disease risk of volunteers. Proc Natl Acad Sci U S A.
110(42):16957-16962.

13. Nontargeted Metabolomics
Broad-spectrum metabolomics platform. 600 metabolites (72 biochemical pathways)
Metabolics has the potential to:
•Detect Metabolic abnormalities that could indicate potential damaging mutations that were previously unappreciated.
•Detect Metabolic signatures consistent with early signs of disease conditions
•Drug effects associated with efficacy and toxicity.

14. Metabolomics diversity of the cohort illustrated by the heat map of the metabolomic profiles
of the volunteers.
Guo L, Milburn MV, Ryals JA, Lonergan SC, Mitchell MW, Wulff JE, Alexander DC, Evans AM, Bridgewater B, Miller L, Gonzalez-
Garay ML, Caskey CT. PNAS 2015;112:E4901-E4910
©2015 by National Academy of Sciences

15. Work flow for searching WES data and metabolomics convergence.
©2015 by National Academy of Sciences
Guo L, Milburn MV, Ryals JA, Lonergan SC, Mitchell MW, Wulff JE, Alexander DC, Evans AM, Bridgewater B, Miller L, Gonzalez-
Garay ML, Caskey CT. PNAS 2015;112:E4901-E4910

16. Plasma metabolomic profiles enhance precision medicine
for volunteers of normal health
No metabolic deficiencies in pathway Metabolic deficiencies in pathway
Guo L, Milburn MV, Ryals JA, Lonergan SC, Mitchell MW, Wulff JE, Alexander DC,
Evans AM, Bridgewater B, Miller L, Gonzalez-Garay ML, Caskey CT. PNAS
2015;112:E4901-E4910

17. Whole Genome Sequence
Family & Medical History
Physical examination
Treatments
Bioinformatics
Interpretation
Metabolomics
Proteomics
Transcriptomics
Modified from Gonzalez-Garay
Personalized Medicine. 2014. 11(05)
The Future of Precision Medicine

18. Bottleneck for precision medicine
1. Genetics and Genomic education for
patients and medical professionals
2. Standards for Genomic interpretation
3. Phenotype-Genotype
4. Instrumentation to detect and describe
new human phenotypes

19. An international effort towards developing standards for best practices in
analysis, interpretation and reporting of clinical genome sequencing
results in the CLARITY Challenge
Genome Biol. 2014 Mar 25;15(3):R53.
Best practices for studying the genome
2012
32 international groups
23 finished the contest
Centronuclear myopathy Cardiac conduction defects Centronuclear myopathy

20. Bioinformatics Pipeline
From Gonzalez-Garay, M.L.
Personalized Medicine. 2014. 11(05)

21. Bottleneck for precision medicine
1. Genetics and Genomic education for
patients and medical professionals
2. Standards for Genomic interpretation
3. Phenotype-Genotype
4. Instrumentation to detect and describe
new human phenotypes

22. Clinical information in Genome(Mendelian disorders)
Caskey & Gonzalez-Garay. Adult Genetic Risk Screening.
Annual Review of Medicine 2014
Non-Coding RNA
•lncRNA
•snRNA
•snoRNA
•RNase P
•RNase MRP
•TERC
•asRNA
•miRNA
•piRNA
•siRNA
•Retrotransposon

23. Rare Genetic Disorders
~7,315 different rare diseases and disorders affecting > 300 million people worldwide
*prevalence of less than 200,000 cases in the USA
∼300 new Mendelian phenotypes are added to OMIM each year

24. Single-Gene Disorders
Mendelian patterns of Inheritance
Dominant
De Novo
RecessiveX-linked recessive
De Novo or recessive
Dominant

25. Phenotype-Genotype and Discovery of Mendelian genes
Chong, J.X., et al. 2015. AJHG 97:2
Loss-of-function mutations

26. Orphan drugs
"Orphan drugs" are medicinal products
intended for diagnosis, prevention or
treatment of life-threatening or debilitating
rare diseases. They are "orphans" because the
pharmaceutical industry has little interest
under normal market conditions in developing
and marketing drugs intended for only a small
number of patients suffering from very rare
conditions.
http://www.eurordis.org/about-orphan-drugs
Medicines in Development
More Than 450 Medicines in Development for
Rare Diseases
http://www.phrma.org/sites/default/files/pdf/Rare_Diseases_2013.pdf

27. Truncating mutations of MAGEL2 cause autism
and Prader-Willi Syndrome-like phenotypes
5 months 13 years
Pervasive Developmental Disorder
•Infantile hypotonia
•Feeding difficulties
•Overweight, but not obese
•Mild intellectual disability
•Autism spectrum disorder
•Methylation testing for PWS
negative
•Negative of Exon Sequencing
•Complete Genomics of the Trio
•A de novo c.1652delT mutation in
MAGEL2 in the PWS critical region.
•Parental studies indicate the
mutation is de novo.
•Due to imprinting, it matters
whether mutation is on the
maternal or paternal allele.
paternal
variant
T G G ? A
maternal
variant
a C A ? T
Well -12,785 -506 -422
deletion
locus
+8,032
inferred fragment
length
F11 + + pat del + > 20 kb
F16 + + pat del + > 20 kb
P23 + + pat del + > 8 kb
I2 + + pat del + > 12 kb
J12 + + pat del + > 20 kb
P4 + + mat T + > 20 kb
C16 - + pat del + > 12 kb
L11 + mat mat T - > 12 kb
I24 mat mat mat T - > 12 kb
K10 mat mat mat T - > 12 kb
D19 - + mat T - > 20 kb
O4 - pat pat del mat > 8 kb
O23 - pat pat del pat > 8 kb
P8 - pat pat del pat > 8 kb
phasing the MAGEL2 mutation

28. Additional patients and phase determination
by methylation sensitive digestion and PCR
Subject Inheritance
Sequence at
mutation site
without digestion
Sequence at
mutation site after
SmaI digestion
Conclusion
1 De novo T/del T
Mutation on pat
allele
2
Not
maternal
C/del C
Mutation on pat
allele
3 De novo AT/del AT
Mutation on pat
allele
4 De novo C/T C
Mutation on pat
allele
c.1652delT first patient
Schaaf CP*#, Gonzalez-Garay M.L.*#, Xia, F*., Potocki, L., Gripp, K. W., Zhang, B., Peters, B. A.,
McElwain, M. A., Drmanac, R., Beaudet, A. L., Caskey, C. T., Yang, Y. (2013)
Truncating mutations of MAGEL2 cause Prader-Willi phenotypes and autism.
Nat Genet. 45:1405-1408.

29. Additional Evidence
•Inactivation of the mouse Magel2 gene results in growth abnormalities similar to
Prader-Willi syndrome.
•Additional patients described by Kingsmore’s group (Sci Transl Med. 2014 Dec)
New phenotype from Prader-Willi Syndrome to SCHAAF-YANG Syndrome
•USP7 is part of the MAGE-L2-TRIM27 ubiquitin ligase and
enables endosomal recycling
•Mutation of USP7 causes a human neurodevelopmental
syndrome, including autism
Hao et al., 2015, Molecular Cell 59, 1–14
September 17, 2015

30. Bottleneck for precision medicine
1. Genetics and Genomic education for
patients and medical professionals
2. Standards for Genomic interpretation
3. Genomic Annotation
4. Instrumentation to detect and describe
new human phenotypes

31. Circulatory systems
cardiovascular system lymphatic system

32. Lymphedema

33. Camera and
intensifier
Laser
controller
785 nm
laser
Data acquisition
and control
system
Intradermal ICG
1.9 mW/cm2
System developed by Dr. Eva Sevick
Near infrared fluorescence lymphatic imaging (NIRFLI)
indocyanine green (ICG)

34. *Gonzalez-Garay, M., *Burrows, P.E., *Rasmussen,
J.C., Aldrich, M.E., Guilloid, R., Maus, E.A., Fife, C.E.,
Kwon., S., Lapinski, P.E., King. P.D., and E.M. Sevick-
Muraca, “Lymphatic abnormalities are associated
with RASA1 mutations in mouse and man,” PNAS,
2013 21;110(21):8621-6
Parkes Weber Syndrome
Capillary malformations and arteriovenous fistulas
Paradominant inheritance model

35. Dilated but functional lymphatic vessels Normal Control
Lymphatic drainage into groin
and pelvic region
*Gonzalez-Garay, M., *Burrows, P.E., et al 2013. PNAS 110(21):8621-6
Inducible mouse knockout for RASA1
indocyanine green (ICG)
Parkes Weber Syndrome (RASA1)
Near infrared fluorescence lymphatic imaging (NIRFLI)
Dr. King, University of Michigan

36. •Approximately 20 genes have been associated with sporadic and familial
lymphedema.
•Only seven genes are used for clinical diagnosis of hereditary lymphedema: FLT4,
FOXC2, GATA2, GJC2, CCBE1, PTPN14, and SOX18.
•There are only 173 variants published in the literature but 87% of the variants
correspond to only two genes FLT4 and FOXC2.
•Mutations in genes from the RAS/MAPK and the PI3K/AKT pathways explain
more than a quarter of the incidence of hereditary lymphedema.
•There are no genetic markers for secondary lymphedema.
State of the union: Genetics of lymphedema.
Pascal B., et al. J Clin Invest. 2014;124(3):898–904.

37. Phenotype of lymphatic disorders in a large family
Agollah, Gonzalez-Garay, et al.
2014. PLOS One 10;9(11):e112548

38. Src Homology 2-domain containing 5’-inositol phosphatase-2 (SHIP2)
PI3K/AKT pathway

39. SHIP2 is required for in vitro lymphangiogenesis
Control
NT
Pool
#1
#2
#3
#4
MW
SHIP2
Actin
HDLEC
siRNA
ControlSHIP2siRNA
(i) (ii) (iii)
(iv) (v) (vi)
Dr. Agollah

40. SHIP2 is required for in vitro lymphangiogenesis
CollagenFibronectin
SHIP2 siRNAControl
0
5
10
15
20
25
30
35
BSA Collagen Fibronectin
AreaFraction(%)
Control
SHIP2 siRNA
*
SHIP2 siRNAControl48hrmigration8hr;VEGFC
Dr. Agollah

41. Large family with lymphedema.
Index Case
Unique phenotype
•Extensive dermal backflow
•Tortuous vessels in both legs
•Defect in the mechanism of lymph
propulsion in collecting vessels and the
development of lymphatic valves

42. Identification of genetic variant
143
152
148
151
154
146
147
150
149
Shows number of reads and
allele fraction
Shows alignments of reads
Shows transcripts with
open reading frames
Affected
Normal
Manuscript submitted for publication, showing biological evidence of a novel gene

43. Targeted Correction and Restored Function of the CFTR Gene
Targeted Correction and Restored Function of the CFTR Gene
in Cystic Fibrosis Induced Pluripotent Stem Cells. 2015 Apr 14;4(4):569-77 Stem Cell Reports.
collaboration with Dr. Brian Davis
Cystic Fibrosis
Repair iPS

44. The future of Genome Engineering: CRISPR/Cas9 Systems

45. Acknowledgments:
•The Cullen Trust for Higher Education
•The Greater Houston Community Foundation
CMI
Dr. Caskey Dr. Soares Dr. Escamilla Dr. Schaaf Dr. Beaudet Dr. Herbst
Dr. Rojas-Martínez Dr. Ortiz-López Dr. Doris Dr. Davis Dr. Northrup Dr. Kit-Sing Au
Dr. Lorenz